Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors

Purpose IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors.

This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers.

In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group.

This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. Trial registration number Supplementary Information The online version contains supplementary material available at 10.1007/s10815-020-02033-9.

Ovarian tissue cryopreservation , Ovarian tissue transplantation , Assisted reproductive technology , Infertility , Pregnancy , Live birth

Every year, many women of childbearing age are treated for cancer by chemotherapy, radiotherapy, and surgery, all of which may affect fertility [1]. Ovarian tissue cryopreservation (OTC) followed by ovarian tissue transplantation (OTT) is one option for fertility restoration in these women, particularly if highly gonadotoxic treatment cannot be delayed or chemotherapy has already begun [2]. The first live birth resulting from a spontaneous pregnancy after such treatment was reported in 2004 [3]. More than 130 live births have since been described in this context [4]. The first live birth achieved after OTT and assisted reproductive technology (ART) management was reported in 2005 [5]. There have been only a few reports of ART use after OTT since, mostly case reports [6–11]. Four case series have been published on this specific topic [12–15]. They enrolled 4, 10, 14, and 28 patients and led to 0, 2, 6, and 7 births, respectively. In 2019, a systematic review on 40 women reported 22 live births [16]. Many reports lack detailed data on ART management, concerning the ovarian stimulation protocol, the number of cycles initiated or of oocyte retrieval procedures performed, the number of oocytes retrieved and their quality, or the number of embryos obtained. Furthermore, the results of fertility tests, including sperm parameters, are not fully described in these main case series. The objectives of this study were to evaluate the efficacy of ART and to describe our experience and the outcomes, spontaneous conceptions and live births in this specific population with or without infertility factors. Patients and methods Study population From 2005 to 2017, 40 patients who underwent ovarian tissue cryopreservation before gonadotoxic treatments were included in a research protocol for OTT (NCT 02184806). These patients included 11 who had failed to conceive spontaneously after OTT, had undergone heterotopic ovarian transplantation, or had associated infertility factors (Fig. 1). These patients were included in this study and began IVF treatment. Seven patients had been treated for hematologic malignancies (5 Hodgkin lymphomas, 2 cases of non-Hodgkin disease), one for invasive cervical carcinoma, one for pseudomyxoma peritonei, one for ovarian borderline tumor, and one for sickle cell disease. OTC was performed between 2000 and 2012, before chemotherapy in five patients and after first-line chemotherapy in six patients who required more gonadotoxic chemotherapy due to disease progression or relapse (Table 1). Table 1. | Patient | Disease | Age at OTC (years) | Chemotherapy before OTC | Type of OTT | Age at OTT (years) | Time from OTT to OFR (months) | Time from OFR to ART (months) | Female infertility factor | Male infertility factor | AFC before ART | FSH (IU/L) before ART | AMH (pmol/L) before ART | |---|---|---|---|---|---|---|---|---|---|---|---|---| | No infertility factors | |||||||||||| | 1 | HL | 31.5 | EBVPx6 | Orthotopic | 36.5 | 3.3 | 11.7 | No | No | 4 | 6.7 | 5.7 | | Orthotopic | 39.7 | - | |||||||||| | 2 | HL | 19.9 | EBVPx8 | Heterotopic | 24.1 | 4.7 | 0.3 | No | No | 10 | 7.1 | 9.3 | | MOPPx3 | Orthotopic | 31.3 | - | ||||||||| | 3 | HL | 27.9 | ABVDx4 | Orthotopic | 35.5 | 4.8 | 3.2 | No | No | 13 | 8 | 2.4 | | Orthotopic | 42.4 | - | |||||||||| | 4 | NHD | 21.3 | CHOPx1 | Orthotopic | 27.2 | 2.2 - | 6.8 | No | No | 6 | 7.9 | < 0.1 | | 5 | NHD | 34.4 | – | Orthotopic | 36.5 | 4.5 - | 2.5 | No | No | 4 | 18 | < 0.1 | | 6 | Sickle cell disease | 21.5 | – | Orthotopic | 33.9 | 4.3 | 10.7 | No | No | 6 | 14 | 5.2 | | Orthotopic | 36.5 | – | |||||||||| | 7 | HL | 18.9 | ABVDx1 | Orthotopic | 26.7 | 3.9 | 33.1 | No | No | 2 | 20.3 | 0.9 | | Infertility factors | |||||||||||| | 8 | HL | 21 | ABVDx8 | Orthotopic | 28.6 | 3.8 | 5.2 | Mild endometriosis | Asthenoteratospermia | 3 | 20 | 3.6 | | 9 | Pseudo-myxoma peritonei | 31.3 | – | Heterotopic | 36.3 | 6 - | 3 | Bilateral adnexectomy | No | 3 | 9 | 3 | | 10 | Invasive cervical carcinoma | 34.7 | – | Orthotopic | 41.9 | 3.8 | 13.2 | Cervix stenosis, thin endometrium, left salpingectomy | Teratospermia | 6 | 26 | 0.1 | | Orthotopic | 44.3 | - | |||||||||| | 11 | Ovarian borderline tumor | 27.1 | – | Orthotopic | 31.8 | 9.1 - | 2.9 | Bilateral adnexectomy | Asthenospermia | 5 | 4.5 | 1.8 | HL Hodgkin lymphoma, NHD non-Hodgkin disease, OTC ovarian tissue cryopreservation, OTT ovarian tissue transplantation, OFR ovarian function recovery ART assisted reproductive technologies Chemotherapy regimens: ABVD: doxorubicin, bleomycin, vinblastine, dacarbazine; CHOP: prednisone, vincristine, cyclophosphamide, doxorubicin; EBVP: epirubicin, bleomycin, vinblastine, prednisone; MOPP: mechlorethamine, vincristine, procarbazine, prednisone Ovarian tissue retrieval and cryopreservation Ovarian tissue was retrieved by unilateral laparoscopic ovariectomy (8 patients) or the removal of ovarian tissue fragments (3 patients). After collection, ovarian tissue was transferred in a sterile transport medium to the laboratory, on ice. The ovarian cortex was isolated from the medulla and was split into fragments. Dimethysulfoxide (DMSO) was used as cryoprotectant. Ovarian tissue was cryopreserved with a slow freezing protocol, as previously described [17], and stored at the temperature of liquid nitrogen. Pre-transplant assessment, thawing, and transplantation of ovarian tissue Before transplantation, a fragment of thawed ovarian tissue was randomly tested for histological evaluation (the ovary fragment was fixed in formaldehyde, embedded in paraffin, cut and stained with hematoxylin-eosin-saffron), and the freezing solution was subjected to bacteriological tests. Premature ovarian failure was confirmed by amenorrhea and determinations of FSH, LH, estradiol, and anti-Müllerian hormone (AMH) levels. A sperm analysis was requested for the woman’s partner. The approval of the treating oncologist/hematologist was obtained before OTT. OTT was performed at a single center, with a one-step procedure. The ovarian fragments were thawed rapidly, as previously described [18]. The thawed tissue was either orthotopically grafted, into peritoneal pockets within the pelvis [19], or heterotopically grafted, into the subcutaneous space of the abdominal wall. During orthotopic ovarian tissue transplantation, the patency of the fallopian tubes was tested. Follow-up and ART After transplantation, ovarian function was monitored monthly by pelvic sonographic and hormonal assessments (FSH, LH, E2, and AMH). Ovarian function recovery (OFR) was defined as the recurrence of spontaneous menstruation. Our strategy involved the initiation of ART soon after ovarian function recovery (OFR) in case of associated infertility factors and heterotopic ovarian tissue transplantation or if natural conception was not achieved within 12 months. ART was performed at six different IVF centers (Poissy-Saint Germain Hospital, Amiens Hospital, Brest Hospital, Nantes Hospital, Tenon Hospital, Les Bluets Hospital). The choice of ovarian stimulation protocol was left to the treating fertility specialist. Data for stimulation protocols, the number of oocytes retrieved, the number and quality of embryos transferred (type A: no or 25%), pregnancies, and live births were reported to the main investigator (CP).

Characteristics of the patients and ART indications (Table 1) The mean age of the patients at OTC was 26.3 ± 6.0 years. Before OTT, all patients had amenorrhea and a hormone profile consistent with premature ovarian failure. In total, 16 OTTs were performed (13 orthotopically in 9 patients; 1 heterotopically for patient 9, because of a medical contraindication to orthotopic OTT; and both heterotopically and orthotopically in patient 2 for a laparoscopy contraindication, which was overcome for her second OTT). Five of the 11 patients underwent two transplantation procedures because of significant reduction of transplant function. The first OTT was performed a mean of 6.3 ± 2.7 years after OTC, at a mean age of 32.6 years ± 5.4 years. The second OTT was performed at a mean age of 38.8 ± 5.1 years. Ovarian function was restored a mean of 4.6 ± 1.8 months after the first OTT for the general population (and a mean of 5.7 ± 2.5 months and 4 ± 0.9 months respectively for the patients with and without associated infertility factors). Fertility test results were abnormal for four couples: three couples had both male and female infertility factors, and one had tubal infertility. The remaining seven couples had no infertility factors. The patients of our cohort underwent their first IVF treatment a mean of 8.4 ± 9.2 months after OFR. In couples with no associated infertility factors, patients underwent their first ART protocol a mean of 9.8 ± 11.1 months after OFR, versus 6.1 ± 4.9 months for couples with at least one associated infertility factor. Patients 10 and 7 had family problems preventing rapid management. Ovarian stimulation protocols (Table 2) Table 2. | Daily dose of gonadotropin | Patients (n) | Initiated cycles (n) | Canceled cycles (n) | Oocytes retrieved (n) | Mean number of oocytes retrieved per cycle initiated (n) | Embryos transferred (n) | |---|---|---|---|---|---|---| | Natural cycle | 1 | 3 | 1 | 2 | 0.7 | 0 | | Modified natural cycle | 2 | 7 | 5 | 1 | 0.1 | 0 | | ≥ 100 to < 225 IU | 5 | 19 | 4 | 16 | 0.8 | 8 | | ≥ 225 to < 300 IU | 5 | 8 | 4 | 12 | 1.5 | 5 | | ≥ 300 IU | 9 | 41 | 9 | 49 | 1.2 | 19 | | Unknown | 4 | 7 | 2 | 15 | 2.1 | 3 | | Total | NA | 85 | 25 | 95 | 1.1 | 35 | NA not applicable In total, 85 cycles were initiated and 60 oocyte retrieval procedures were performed. Forty-three cycles were initiated with antagonist protocols, 13 with short agonist protocols, and 12 with long agonist protocols; seven were modified natural cycles and three were natural cycles. In seven cases, details about the type of stimulation protocol were missing. Descriptions of each stimulation protocol are added in Online Resource 1. Nine of the 11 patients underwent variable protocols, with variable daily doses of gonadotropins; one patient (patient 7) underwent only one type of stimulation protocol (two long agonist protocols with high daily doses of gonadotropins); and one patient (patient 4) underwent stimulation only once. Recombinant FSH or human menopausal gonadotropin (hMG) was used for stimulation. In the group with infertility factors, the mean number of cycles initiated per patient was 11 ± 13, with a median of 6 cycles per patient (range 2–30); in the group without infertility factors, the mean number of cycles initiated per patient was 5.9 ± 5.7, with a median of 3 cycles per patient (range 1–14). Twenty-five cycles (29.4%) were canceled due to a lack of ovarian response (9 cycles) or premature ovulation before introduction of LHRH antagonist and/or impossibility to switch from IVF to IUI due to oligoasthenoteratospermia or bilateral annexectomy (16 cycles). All types of stimulation protocol were concerned, but particularly natural and modified natural cycles (60%). Almost half the cycles with no ovarian response occurred in patient 11, despite the use of different types of stimulation protocol. None of the patients presented OHSS (ovarian hyperstimulation syndrome). Patient 2 had a transient ischemic attack after her last IVF attempt. Oocytes, fertilization rate, and embryos (Table 3) Table 3. | Patient | Number of stimulation cycles/number of oocyte retrievals | Number of natural or modified natural cycles/number of oocyte retrievals | Total number of oocytes retrieved | Total number of metaphase II oocytes | Total number of zygotes | Total number of embryos transferred | Pregnancies/live births after ART | Spontaneous pregnancies/live births | |---|---|---|---|---|---|---|---|---| | No infertility factors | |||||||| | 1 | 3/3 | 0 | 3 | 3 | 3 | 2 | 0 | 3/1 | | 2 | 12/12 | 2/2 | 30 | 10 | 6 | 6 | 0 | 0/0 | | 3 | 2/2 | 0 | 7 | 7 | 5 | 2 | 0 | 3/1 | | 4 | 1/0 | 0 | 0 | 0 | 0 | 0 | 0 | 1/1 | | 5 | 13/10 | 1/0 | 11 | 7 | 5 | 4 | 0 | 1*/− | | 6 | 5/3 | 0 | 2 | 1 | 0 | 0 | 0 | 0/0 | | 7 | 2/2 | 0 | 5 | 4 | 4 | 2 | 0 | 1/1 | | Infertility factors | |||||||| | 8 | 28/21 | 2/0 | 32 | 25 | 18 | 18 | 2/1 | 0/0 | | 9 | 2/2 | 0 | 2 | 0 | 0 | 0 | 0 | 0/0 | | 10 | 3/0 | 0 | 0 | 0 | 0 | 0 | 0 | 0/0 | | 11 | 4/1 | 5/2 | 3 | 2 | 1 | 1 | 0 | 0/0 | | Total | 75/56 | 10/4 | 95 | 59 | 42 | 35 | 2/1 | 8/4 | *Ongoing pregnancy We found that 36 of the 95 oocytes retrieved were abnormal or immature (37.9%). Sixteen oocyte retrieval procedures revealed empty follicles (16/60 = 26.7%). The mean number of oocytes retrieved was 1.1 per initiated cycle and 1.6 (range 0–9) per oocyte retrieval procedure. The mean number of oocytes retrieved per cycle initiated was 0.7 for natural cycles, 0.1 for modified natural cycles, 0.8 for protocols with a low daily dose of gonadotropin (100 ≤ dose <225 IU), 1.5 for protocols with an intermediate daily dose of gonadotropin (225 ≤ dose <300 IU), and 1.2 for protocols with a high daily dose of gonadotropin (≥ 300 IU). Forty-two zygotes were obtained: the fertilization rate was 74% after 23 ICSI procedures and 65% after 10 IVF procedures. Four embryos from two patients were cryopreserved: three for patient 8 and one for patient 3. Finally, 35 embryos from 27 cycles were transferred at day 2 or 3 into seven women (5 patients without infertility factor and 2 patients with infertility factor): two embryos were transferred for eight cycles because one of the embryo had a lower quality grade (this was clinic protocol for day 2–3 embryo transfer if one of the embryo had a lower quality grade), and one embryo was transferred for 19 cycles (including the transfer of three frozen embryos after hormone replacement therapy cycle). Fifty-seven point one percent of the embryos were type A, 8.6% type B, 22.9% type C, and for the remaining 11.4%, the data were not available. Oocyte retrieval was not possible in patients 4 and 10, due to premature ovulation. No embryos were obtained for two patients: patient 6, who underwent five stimulations, with the retrieval of one mature oocyte but unsuccessful fertilization, and patient 9, who underwent heterotopic transplantation, with no mature oocytes obtained. Pregnancies and live births after ART and spontaneous conception (Table 3) One patient with infertility factors (patient 8) had two pregnancies resulting from ART, one ending in an early miscarriage and the other in a live birth. A healthy boy, measuring 47 cm and weighing 2330 g, was born by vaginal delivery at 37 weeks and 6 days of gestation. Pregnancy and live birth rates were 2.4% and 1.2% per cycle initiated, respectively, and 7.4% and 3.7% per embryo transfer, respectively. For the seven patients who had embryos transferred, the rates of pregnancy and live birth were 28.6% and 14.3% per patient, respectively. No pregnancy was obtained after frozen embryo transfer. Five patients conceived spontaneously after the failure of IVF treatment (patients 1, 3, 4, 5, and 7): nine pregnancies were obtained, leading to four live births, three miscarriages, and one abortion for personal reasons. A pregnancy is still ongoing. The time from the last IVF cycle to the start of the spontaneous pregnancy was 10 months for patient 1, 22 months for patient 3, 3 months for patient 4, 39 months for patient 5, and 6 months for patient 7. Two patients conceived three times (patients 1 and 3). All these women who conceived spontaneously had no associated infertility factors. In our cohort, 9.1% of women had at least one live-born baby they could take home after ART versus 36.4% of women after spontaneous conception.

Our results show that ART treatments in women with grafted frozen-thawed ovarian tissue are associated with a high risk of canceled cycles, empty follicles, abnormal or immature oocytes, and low subsequent pregnancy and live birth rates. However, this specific population had a high rate of spontaneous pregnancies in women without associated infertility factors. This study reports the rate of canceled cycles in this situation and expresses the pregnancy and birth rates per initiated cycle, providing information about the real efficacy of this treatment. Nevertheless, this cohort is still rather small. Our study is the first one to fully describe fertility tests prior to ART management. In Table 4, we show the results for our cohort and those for the four principal studies previously published. In our study, mean age at OTC (26.3 ± 6.0 years) and OTT (32.6 ± 5.4 years) were similar to those calculated for 40 patients in a previous review (26.8 ± 6.8 years and 32.3 ± 6.3 years, respectively) [16]. In the absence of fertility abnormalities, there is no consensus concerning the most appropriate timing of ART after OTT: Meirow et al. enrolled patients directly after OFR, Dolmans et al. and Schmidt et al. waited up to 1 year for natural conception, and Dueholm Hjorth et al. waited 6 months [12–15]. In the review by Andersen et al., most women (80%) began IVF treatment directly after OFR [16]. In the subgroup with no associated infertility factors, we agreed to IVF treatment without delay in three patients: patients 2 and 3 were the first two patients to undergo transplantation, at a time at which we had a more interventionist management strategy, and patient 5 requested IVF because of her history of unexplained subfertility, with a previous spontaneous pregnancy ending in an elective abortion due to a polymalformative syndrome. Patient 7 separated from her first partner after OTT and was single for several years. In the subgroup with associated infertility factors, patient 10 was lost to follow-up for a few months due to family problems, increasing the time to IVF treatment initiation. Table 4. | Study | Dolmans et al. [12] | Schmidt et al. [13] | Meirow et al. [14] | Dueholm Hjorth et al. [15] | Our study | |---|---|---|---|---|---| | Patients (n) | 4 | 10 | 14 | 28 | 11 | | Mean age ± SD at OTC (years) | 23.5 ± 3.1 | 28.3 ± 5.1 | 28.7 ± 7.3 | 29.8 ± 5.2 | 26.3 ± 6 | | Mean age ± SD at OTT (years) | NA | NA | 34.1 ± 5.9 | 34 ± 5.1 | 32.6 ± 5.4 | | OFR to ART (months) | 12 | After natural conception attempted | Soon after OFR | NA | 8.4 ± 9.2 (mean ± SD) | | Associated infertility factors | Not specified but normal sperm parameters | NA | NA | NA | Available | | ART cycles (n) | 21 | 56 | 56 | 99 | 85 | | Mean number of cycles/patient | 5.2 (range 4–7) | 5.6 (range 1–12) | 4 (range 1–11) | 3.5 (range 1–14) | 7.7 (range 1–30) | | Ovarian stimulation protocol | 13 MNC + 8 NC | Mostly MNC | Mostly MNC | Mostly antagonist (68.7%) and long agonist (29.3%) | Mostly antagonist and high daily dose of gonadotropins | | Cancelation rate (%) | NA | NA | NA | NA | 29.4 | | Retrieved oocytes (n) | 16 | 49 | 84 | 193 | 95 | | MII (n) | 10 | 33 | 84* | 193* | 59 | | Empty follicle rate (%) | 29 | NA | 23.5 | 35.8 | 26.6 | | Mean number of oocytes/cycle initiated | 0.8 (range 0–2) | 0.9 (range NA) | 1.5 (range 0–5) | 1.95 (range 0–14) | 1.1 (range 0–9) | | ART procedure | ICSI | IVF | IVF | ICSI | IVF (n = 10) and ICSI (n = 23) | | Fertilization rate per MII (%) | 50 | 54.5 | 58 | 38 | IVF 65, ICSI 74.3 | | Transferred embryos (n) | 5 | 16 | 38 | 46 | 35 | | Embryo transfers (n) | 5 | 15 | NA | 36 | 27 | | Pregnancies after ART (n) | 0 | 5 | 10 | 15 | 2 | | Pregnancy rate/transfer (%) | 0 | 33.3 | 26 | 41.6 | 7.4 | | Live births after ART (n) | 0 | 2 | 6 (+ 1 ongoing pregnancy) | 7 | 1 | | Live birth rate/cycles initiated (%) | 0 | 3.6 | 10.7 | 7.1 | 1.2 | | Live birth rate/transfers (%) | 0 | 13.3 | 16 | 19.4 | 3.7 | | Spontaneous pregnancies (n) | NA | 1 | 6 | 1 | 8 (+ 1 ongoing pregnancy) | | Spontaneous live births (n) | NA | 1 | 3 | 1 | 4 | | Percentage of patients with at least one child | NA | 20 | 35.7 | 21.4 | 45.5 | NA not available, MNC modified natural cycle, NC natural cycle, MII metaphase II oocyte, NR not reported, PR pregnancy rate *Number of MII not indicated In our cohort, the mean number of cycles initiated per patient was 5.5 ± 5 if patient 8, who underwent 30 cycles, was excluded from the analysis, and this number is consistent with the four main published cohort studies, which reported a mean number of cycles between 3.5 and 5.6 (Table 4). Furthermore, our study is the only one to detail the number of cycles initiated according to the presence or absence of infertility factors, with more cycles performed in the subgroup with infertility factors. In our study, different types of ovarian stimulation protocols were used between and within IVF centers, and also within individual patients. Indeed, this was a multicenter study and the choice of stimulation protocol was left to the discretion of the treating fertility specialist. In our cohort, antagonist protocols were generally preferred, as Dueholm Hjorth et al. [15], whereas natural and modified natural cycles predominated in the three other main cohort studies [12–14]. A recent review identified no significant difference in reproductive outcome between modified natural cycles and antagonist protocols [16]. In our study, the median number of mature oocytes retrieved per oocyte retrieval procedure (1, range 0–5) was similar to that reported in the review by Andersen et al. (1, range 0–8) [16]. Cycles with intermediate daily doses of gonadotropin (≥ 225 to < 300 IU per day) gave the highest oocyte yields in our study, but this result must be interpreted with caution because the number of cycles is low and the cumulative dose of gonadotropin per cycle is unknown. Surprisingly, the mean number of oocytes retrieved per retrieval procedure was 1.6, using mostly antagonist protocols and high daily doses of gonadotropins, whereas Meirow et al. retrieved almost the same mean number of oocytes per oocyte retrieval (1.5) mostly with modified natural cycles [14]. One reason for this may be the timing of ART initiation, with better results obtained if IVF treatment is initiated soon after OFR because of the higher follicular density in the transplants and initial follicular growth activation. This finding also suggests that high doses of gonadotropin are probably not necessary. Nevertheless, for women predicted to be poor responders, it remains unclear whether a natural or modified natural IVF cycle is better than conventional stimulation, and no difference in live birth/ongoing pregnancy rates were observed with gonadotropin doses of more than 150 IU [20]. The rates of empty follicles and immature-abnormal oocytes were 26.7% and 37.9%, respectively, and these values are similar to published results for this specific population [12, 14, 15]. Several mechanisms have been put forward to explain these phenomena: dysfunctional folliculogenesis, early oocyte atresia with an apparently normal hormone response, asynchrony between granulosa cell and oocyte maturation, or damage to the oocyte itself as a result of freezing, thawing, and transplantation [12, 21]. In this study, the ART used was decided on the basis of sperm parameters, except for patients 10 and 7, for whom ICSI was performed regardless of the normality of sperm parameters, in accordance with the IVF center’s strategy for an expected weak ovarian response. Our fertilization rate is higher than those of the four main published studies. Nevertheless, despite high rates of fertilization and the transfer of relatively large numbers of embryos, we obtained a lower rate of live births per cycle initiated (1.2%) than three other main cohorts [13–15] and the recent review including 40 women (10.3%) [16]. Nevertheless, the rates reported in the literature review by Andersen et al. should be interpreted with caution, as 75% of the articles considered were case reports, which are known to be more likely to present positive than negative results, potentially leading to an overestimation of this outcome [16]. In the study by Schmidt et al., only the number of cycles with oocyte retrieval is given, not the number of cycles initiated [13]. Nevertheless, in the studies by Schmidt et al. and Meirow et al., all patients were managed at a single center, making it possible for the center concerned to gain important experience. This was not the case in our study, in which each center treated only one to four patients. Furthermore, the center at which the babies were delivered after OTT and IVF also managed the largest number of patients (4), highlighting the importance of experience. In our cohort, the cancelation rate was very high (29.4%). Our study was the second study to express pregnancy and birth rates per initiated cycle, which provides better information about the real efficacy of ART management. In our cohort, four patients without infertility factors gave birth to a healthy child after spontaneous conception. One ongoing pregnancy occurred 4 years and 9 months after OTT and two patients conceived three times. This clearly confirms the long-term survival of the ovarian transplants and their ability to give rise to a pregnancy, even a long time after transplantation [22]. In the four main published cohorts, none, one, three, and one patient conceived naturally [12–15]. In our study, 11 pregnancies were obtained, nine of which in patients without infertility factors. Five live births occurred, 4 of which after spontaneous conception in the group without infertility factors. Finally, the total baby take home rate in our cohort was 45.5%, versus 35.7%, 20%, and 21.4% respectively in the series of Meirow et al., Schmidt et al., and Dueholm Hjorth et al. [13–15]. In conclusion, our study confirms poor reproductive outcomes after ART in women with grafted frozen-thawed ovarian tissue and highlights the high probability of spontaneous pregnancy in this specific population. There is currently no consensus concerning the ART strategy to be used in women who have undergone OTT, in terms of either the timing of ART after transplantation or the ovarian stimulation protocol. Our results indicate that spontaneous fertility strategies may be more appropriate than intensive IVF treatments for couples without associated infertility factors. Personalized strategies should be considered, according to the patient’s age, associated infertility factors, motivations, and the expected life time of the ovarian transplants [18]. Our study also provides additional information on transplanted women with associated infertility factors and no possibility of spontaneous pregnancy. These women should be informed of the low chance of live birth after ART management. High-quality controlled studies are required to improve our knowledge of the most appropriate type of ART management and the most efficient ovarian stimulation protocols in this population, and a large amount of practice seems to be required to improve outcomes. Supplementary Information Acknowledgments We would like to thank the hematologists and surgeons who referred their patients to us: Prof. Sylvie Castaigne, Prof. Alain Delmer, Prof. Bernard Desablens, Prof. Dominique Elias, Dr. Thierry Forge, Dr. Jean Gabarre, Dr. Reda Garidi, Dr. Kamel Ghomari, Dr. Hugo Gonzales, Dr. Catherine Lhommé, Prof. Philippe Merviel, Prof. Regis Peffault de Latour, Prof. Gérard Socié, Dr. Dan Stefanescu, Dr. Aliénor Xhaard. We thank Dr. Hervé Oya for data management, Prof. Alain Mallet, Dr. Merry Mazmanian, Dr. Cécile Kedzia, project managers. We also thank Corinne Journo and Lilia Grira for technical assistance. We thank Prof. Jim Thornton for kindly providing editorial assistance and critically reviewing the manuscript. Authors’ contribution C.P. was responsible for the fertility preservation program and for the ovarian transplantation protocol. M.V. and C.P. designed the study, interpreted the data, and contributed to the writing and revision of the manuscript. M.V. collected all the data and contributed to the preparation of the original draft. B.P., A.T., F.S., MT.leM., F.L., L.S., and A.N. assessed fertility and were responsible for clinical ART after transplantation. M.PB., S.M., and M.B. were responsible for biological ART after ovarian tissue transplantation. V.BL was responsible for the ovarian tissue thawing program. N.B. contributed to data interpretation. A.F. performed the ovarian tissue transplantation procedure. All authors contributed to the writing and revision of the manuscript. Funding The ovarian tissue transplantation research protocol was supported by the “Agence de la Biomedecine” (AOR AMP 2007) (France’s biomedicine agency) and by the “Association Saint Louis,” a patient-funded charity. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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